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In some circumstances, the eukaryotic cell cycle can be best described as a succession of contingent events. For example, in most somatic cells in culture, cell growth is followed by DNA replication, then more cell growth, then mitotic entry and chromosome congression, then sister chromatid separation and mitotic exit. The transitions from one cell cycle phase to the next are generally all-or-none in character and irreversible. Our goal is to understand how these irreversible switches between phases occur.

In other contexts the cell cycle is better described as an autonomous oscillation. For example, in the early Xenopus embryo, every ~30 minutes CDK1 is activated and this reliable rhythm is maintained even if DNA replication or mitosis is blocked. Our goal is to understand how this oscillator works.

The approaches we have taken to these questions include quantitative experimental approaches, computational modeling, and the theory of nonlinear dynamics. We hope to understand the design principles of these systems, and perhaps to gain insight into other biological switches and oscillators as well.

Publications

All Publications

Abstract

Negative cooperativity is a phenomenon in which the binding of one or more molecules of a ligand to a multimeric receptor makes it more difficult for subsequent ligand molecules to bind. Negative cooperativity can make a multimeric receptor's response more graded than it would otherwise be. However, through theory and experimental results, we show that if the ligand binds the receptor with high affinity and can be appreciably depleted by receptor binding, then negative cooperativity produces a qualitatively different type of response: a highly ultrasensitive response with a pronounced threshold. Because ultrasensitivity and thresholds are important for generating various complex systems-level behaviors, including bistability and oscillations, negative cooperativity may be an important ingredient in many types of biological responses.

Abstract

Previous work has shown that Suc1/Cks proteins can promote the hyperphosphorylation of primed Cdk1 substrates through the formation of ternary Cdk1-Cks-phosphosubstrate complexes. This raises the possibility that Cks proteins might be able to both facilitate and interfere with hyperphosphorylation through a mechanism analogous to the prozone effect in antigen-antibody interactions, with substoichiometric Cks promoting the formation of Cdk1-Cks-phosphosubstrate complexes and suprastoichiometric Cks instead promoting the formation of Cdk1-Cks and Cks-phosphosubstrate complexes. We tested this hypothesis through a combination of theory, proof-of-principle experiments with oligonucleotide annealing, and experiments on the interaction of Xenopus cyclin B1-Cdk1-Cks2 with Wee1A in vitro and in Xenopus extracts. Our findings help explain why both Cks under-expression and overexpression interfere with cell-cycle progression and provide insight into the regulation of the Cdk1 system.

Abstract

Theoretical studies have shown that a deterministic biochemical oscillator can become chaotic when operating over a sufficiently large volume and have suggested that the Xenopus laevis cell cycle oscillator operates close to such a chaotic regime. To experimentally test this hypothesis, we decreased the speed of the post-fertilization calcium wave, which had been predicted to generate chaos. However, cell divisions were found to develop normally, and eggs developed into normal tadpoles. Motivated by these experiments, we carried out modeling studies to understand the prerequisites for the predicted spatial chaos. We showed that this type of spatial chaos requires oscillatory reaction dynamics with short pulse duration and postulated that the mitotic exit in Xenopus laevis is likely slow enough to avoid chaos. In systems with shorter pulses, chaos may be an important hazard, as in cardiac arrhythmias, or a useful feature, as in the pigmentation of certain mollusk shells.

Abstract

Trigger waves are a recurring biological phenomenon involved in transmitting information quickly and reliably over large distances. Well-characterized examples include action potentials propagating along the axon of a neuron, calcium waves in various tissues, and mitotic waves in Xenopus eggs. Here we use the FitzHugh-Nagumo model, a simple model inspired by the action potential that is widely used in physics and theoretical biology, to examine different types of trigger waves-spatial switches, pulses, and oscillations-and to show how they arise.

Abstract

In this series of reviews, we are examining ultrasensitive responses, the switch-like input-output relationships that contribute to signal processing in a wide variety of signaling contexts. In the first part of this series, we explored one mechanism for generating ultrasensitivity, zero-order ultrasensitivity, where the saturation of two converting enzymes allows the output to switch from low to high over a tight range of input levels. In this second installment, we focus on three conceptually distinct mechanisms for ultrasensitivity: multisite phosphorylation, stoichiometric inhibitors, and positive feedback. We also examine several related mechanisms and concepts, including cooperativity, reciprocal regulation, coherent feed-forward regulation, and substrate competition, and provide several examples of signaling processes where these mechanisms are known or are suspected to be applicable.

Abstract

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

Abstract

The study of eukaryotic cell cycle regulation over the last several decades has led to a remarkably detailed understanding of the complex regulatory system that drives this fundamental process. This allows us to now look for recurring motifs in the regulatory system. Among these are negative feedback loops, which underpin checkpoints and generate cell cycle oscillations; positive feedback loops, which promote oscillations and make cell cycle transitions switch-like and unidirectional; and reciprocal regulation, which can increase the control a key regulator exerts. These simple motifs are found at multiple points in the cell cycle (e.g. S-phase and M-phase control) and are conserved in diverse organisms. These findings argue for an underlying unity in the principles of cell cycle control.

Abstract

Despite the large size of the Xenopus laevis egg (approximately 1.2 mm diameter), a fertilized egg rapidly proceeds through mitosis in a spatially coordinated fashion. Mitosis is initiated by a bistable system of regulatory proteins centred on Cdk1 (refs 1, 2), raising the possibility that this spatial coordination could be achieved through trigger waves of Cdk1 activity. Using an extract system that performs cell cycles in vitro, here we show that mitosis does spread through Xenopus cytoplasm via trigger waves, propagating at a linear speed of approximately 60 µm min(-1). Perturbing the feedback loops that give rise to the bistability of Cdk1 changes the speed and dynamics of the waves. Time-lapse imaging of intact eggs argues that trigger waves of Cdk1 activation are responsible for surface contraction waves, ripples in the cell cortex that precede cytokinesis. These findings indicate that Cdk1 trigger waves help ensure the spatiotemporal coordination of mitosis in large eggs. Trigger waves may be an important general mechanism for coordinating biochemical events over large distances.

Abstract

Despite the complexity and variety of biological oscillators, their core design invariably includes an essential negative feedback loop. In the Xenopus laevis embryonic cell cycle oscillator, this loop consists of the kinase cyclin B-Cdk1 and the ubiquitin ligase APC/C(Cdc20); active Cdk1 activates APC/C(Cdc20), which then brings about cyclin B degradation and inactivates Cdk1. Here we ask how this negative feedback loop functions quantitatively, with the aim of understanding what mechanisms keep the Cdk1-APC/C(Cdc20) system from settling into a stable steady state with intermediate levels of Cdk1 and APC/C(Cdc20) activity. We found that the system operates as a time-delayed, digital switch, with a time lag of ∼ 15 min between Cdk1 and APC/C(Cdc20) activation and a tremendously high degree of ultrasensitivity (nH≈17). Computational modelling shows how these attributes contribute to the generation of robust, clock-like oscillations. Principles uncovered here may also apply to other activator-repressor oscillators and help in designing robust synthetic clocks.

Abstract

Mitosis is triggered by the activation of Cdk1-cyclin B1 and its translocation from the cytoplasm to the nucleus. Positive feedback loops regulate the activation of Cdk1-cyclin B1 and help make the process irreversible and all-or-none in character. Here we examine whether an analogous process, spatial positive feedback, regulates Cdk1-cyclin B1 redistribution. We used chemical biology approaches and live-cell microscopy to show that nuclear Cdk1-cyclin B1 promotes the translocation of Cdk1-cyclin B1 to the nucleus. Mechanistic studies suggest that cyclin B1 phosphorylation promotes nuclear translocation and, conversely, nuclear translocation promotes cyclin B1 phosphorylation, accounting for the feedback. Interfering with the abruptness of Cdk1-cyclin B1 translocation affects the timing and synchronicity of subsequent mitotic events, underscoring the functional importance of this feedback. We propose that spatial positive feedback ensures a rapid, complete, robust, and irreversible transition from interphase to mitosis and suggest that bistable spatiotemporal switches may be widespread in biological regulation.

Abstract

Waddington's epigenetic landscape is probably the most famous and most powerful metaphor in developmental biology. Cells, represented by balls, roll downhill through a landscape of bifurcating valleys. Each new valley represents a possible cell fate and the ridges between the valleys maintain the cell fate once it has been chosen. Here I examine models of two important developmental processes - cell-fate induction and lateral inhibition - and ask whether the landscapes for these models at least qualitatively resemble Waddington's picture. For cell-fate induction, the answer is no. The commitment of a cell to a new fate corresponds to the disappearance of a valley from the landscape, not the splitting of one valley into two, and it occurs through a type of bifurcation - a saddle-node bifurcation - that possesses an intrinsic irreversibility that is missing from Waddington's picture. Lateral inhibition, a symmetrical cell-cell competition process, corresponds better to Waddington's picture, with one valley reversibly splitting into two through a pitchfork bifurcation. I propose an alternative epigenetic landscape that has numerous valleys and ridges right from the start, with the process of cell-fate commitment corresponding to the irreversible disappearance of some of these valleys and ridges, via cell-fate induction, complemented by the creation of new valleys and ridges through processes like cell-cell competition.

Abstract

When several genes or proteins modulate one another's activity as part of a network, they sometimes produce behaviors that no protein could accomplish on its own. Intuition for these emergent behaviors often cannot be obtained simply by tracing causality through the network in discreet steps. Specifically, when a network contains a feedback loop, biologists need specialized tools to understand the network's behaviors and their necessary conditions. This analysis is grounded in the mathematics of ordinary differential equations. We, however, will demonstrate the use of purely graphical methods to determine, for experimental data, the plausibility of two network behaviors, bistability and irreversibility. We use the Xenopus laevis oocyte maturation network as our example, and we make special use of iterative stability analysis, a graphical tool for determining stability in two dimensions.

Abstract

Protein phosphorylation provides a mechanism for the rapid, reversible control of protein function. Phosphorylation adds negative charge to amino acid side chains, and negatively charged amino acids (Asp/Glu) can sometimes mimic the phosphorylated state of a protein. Using a comparative genomics approach, we show that nature also employs this trick in reverse by evolving serine, threonine, and tyrosine phosphorylation sites from Asp/Glu residues. Structures of three proteins where phosphosites evolved from acidic residues (DNA topoisomerase II, enolase, and C-Raf) show that the relevant acidic residues are present in salt bridges with conserved basic residues, and that phosphorylation has the potential to conditionally restore the salt bridges. The evolution of phosphorylation sites from glutamate and aspartate provides a rationale for why phosphorylation sometimes activates proteins, and helps explain the origins of this important and complex process.

Abstract

Computational modeling and the theory of nonlinear dynamical systems allow one to not simply describe the events of the cell cycle, but also to understand why these events occur, just as the theory of gravitation allows one to understand why cannonballs fly in parabolic arcs. The simplest examples of the eukaryotic cell cycle operate like autonomous oscillators. Here, we present the basic theory of oscillatory biochemical circuits in the context of the Xenopus embryonic cell cycle. We examine Boolean models, delay differential equation models, and especially ordinary differential equation (ODE) models. For ODE models, we explore what it takes to get oscillations out of two simple types of circuits (negative feedback loops and coupled positive and negative feedback loops). Finally, we review the procedures of linear stability analysis, which allow one to determine whether a given ODE model and a particular set of kinetic parameters will produce oscillations.

Abstract

Cdc25C is a critical component of the interlinked positive and double-negative feedback loops that constitute the bistable mitotic trigger. Computational studies have indicated that the trigger's bistability should be more robust if the individual legs of the loops exhibit ultrasensitive responses. Here, we show that in Xenopus extracts two measures of Cdc25C activation (hyperphosphorylation and Ser 287 dephosphorylation) are highly ultrasensitive functions of the Cdk1 activity; estimated Hill coefficients were 11 to 32. Some of Cdc25C's ultrasensitivity can be reconstituted in vitro with purified components, and the reconstituted ultrasensitivity depends upon multisite phosphorylation. The response functions determined here for Cdc25C and previously for Wee1A allow us to formulate a simple mathematical model of the transition between interphase and mitosis. The model shows how the continuously variable regulators of mitosis work collectively to generate a switch-like, hysteretic response.

The Roles of Cyclin A2, B1, and B2 in Early and Late Mitotic EventsMOLECULAR BIOLOGY OF THE CELLGong, D., Ferrell, J. E.2010; 21 (18): 3149-3161

Abstract

Here we have used siRNAs and time-lapse epifluorescence microscopy to examine the roles of various candidate mitotic cyclins in chromatin condensation in HeLa cells. Knocking down cyclin A2 resulted in a substantial (?7 h) delay in chromatin condensation and histone H3 phosphorylation, and expressing an siRNA-resistant form of cyclin A2 partially rescued chromatin condensation. There was no detectable delay in DNA replication in the cyclin A2 knockdowns, arguing that the delay in chromatin condensation is not secondary to a delay in S-phase completion. Cyclin A2 is required for the activation and nuclear accumulation of cyclin B1-Cdk1, raising the possibility that cyclin B1-Cdk1 mediates the effects of cyclin A2. Consistent with this possibility, we found that chromatin condensation was tightly associated temporally with the redistribution of cyclin B1 to the nucleus. Moreover, a constitutively nuclear cyclin B1 rescued chromatin condensation in cyclin A2 knockdown cells. On the other hand, knocking down cyclin B1 delayed chromatin condensation by only about one hour. Our working hypothesis is that active, nuclear cyclin B1-Cdk1 normally cooperates with cyclin A2 to bring about early mitotic events. Because cyclin A2 is present only during the early stages of mitosis, we asked whether cyclin B knockdown might have more dramatic defects on late mitotic events. Consistent with this possibility, we found that cyclin B1- and cyclin B1/B2-knockdown cells had difficulty in maintaining a mitotic arrest in the presence of nocodazole. Taken together, these data suggest that cyclin A2 helps initiate mitosis, in part through its effects on cyclin B1, and that cyclins B1 and B2 are particularly critical for the maintenance of the mitotic state.

Abstract

Systems biology takes an interdisciplinary approach to the systematic study of complex interactions in biological systems. This approach seeks to decipher the emergent behaviors of complex systems rather than focusing only on their constituent properties. As an increasing number of examples illustrate the value of systems biology approaches to understand the initiation, progression, and treatment of cancer, systems biologists from across Europe and the United States hope for changes in the way their field is currently perceived among cancer researchers. In a recent EU-US workshop, supported by the European Commission, the German Federal Ministry for Education and Research, and the National Cancer Institute of the NIH, the participants discussed the strengths, weaknesses, hurdles, and opportunities in cancer systems biology.

Abstract

Here we review some of our work over the last decade on Xenopus oocyte maturation, a cell fate switch, and the Xenopus embryonic cell cycle, a highly dynamical process. Our approach has been to start with wiring diagrams for the regulatory networks that underpin the processes; carry out quantitative experiments to describe the response functions for individual legs of the networks; and then construct simple analytical models based on chemical kinetic theory and the graphical rate-balance formalism. These studies support the view that the all-or-none, irreversible nature of oocyte maturation arises from a saddle-node bifurcation in the regulatory system that drives the process, and that the clock-like oscillations of the embryo are built upon a hysteretic switch with two saddle-node bifurcations. We believe that this type of reductionistic systems biology holds great promise for understanding complicated biochemical processes in simpler terms.

Abstract

New experimental and theoretical studies reported by Uri Alon, Marc Kirschner, and colleagues in this issue of Molecular Cell suggest that Weber's law of sensory perception may apply to a number of cell signaling processes.

Abstract

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.

Abstract

Determining proper responsiveness to incoming signals is fundamental to all biological systems. We demonstrate that intracellular signaling nodes can tune a signaling network's response threshold away from the basal median effective concentration established by ligand-receptor interactions. Focusing on the bistable kinase network that governs progesterone-induced meiotic entry in Xenopus oocytes, we characterized glycogen synthase kinase-3beta (GSK-3beta) as a dampener of progesterone responsiveness. GSK-3beta engages the meiotic kinase network through a double-negative feedback loop; this specific feedback architecture raises the progesterone threshold in correspondence with the strength of double-negative signaling. We also identified a marker of nutritional status, l-leucine, which lowers the progesterone threshold, indicating that oocytes integrate additional signals into their cell-fate decisions by modulating progesterone responsiveness.

Abstract

The main conclusion is that systems biology approaches can indeed advance cancer research, having already proved successful in a very wide variety of cancer-related areas, and are likely to prove superior to many current research strategies. Major points include: Systems biology and computational approaches can make important contributions to research and development in key clinical aspects of cancer and of cancer treatment, and should be developed for understanding and application to diagnosis, biomarkers, cancer progression, drug development and treatment strategies. Development of new measurement technologies is central to successful systems approaches, and should be strongly encouraged. The systems view of disease combined with these new technologies and novel computational tools will over the next 5-20 years lead to medicine that is predictive, personalized, preventive and participatory (P4 medicine).Major initiatives are in progress to gather extremely wide ranges of data for both somatic and germ-line genetic variations, as well as gene, transcript, protein and metabolite expression profiles that are cancer-relevant. Electronic databases and repositories play a central role to store and analyze these data. These resources need to be developed and sustained. Understanding cellular pathways is crucial in cancer research, and these pathways need to be considered in the context of the progression of cancer at various stages. At all stages of cancer progression, major areas require modelling via systems and developmental biology methods including immune system reactions, angiogenesis and tumour progression.A number of mathematical models of an analytical or computational nature have been developed that can give detailed insights into the dynamics of cancer-relevant systems. These models should be further integrated across multiple levels of biological organization in conjunction with analysis of laboratory and clinical data.Biomarkers represent major tools in determining the presence of cancer, its progression and the responses to treatments. There is a need for sets of high-quality annotated clinical samples, enabling comparisons across different diseases and the quantitative simulation of major pathways leading to biomarker development and analysis of drug effects.Education is recognized as a key component in the success of any systems biology programme, especially for applications to cancer research. It is recognized that a balance needs to be found between the need to be interdisciplinary and the necessity of having extensive specialist knowledge in particular areas.A proposal from this workshop is to explore one or more types of cancer over the full scale of their progression, for example glioblastoma or colon cancer. Such an exemplar project would require all the experimental and computational tools available for the generation and analysis of quantitative data over the entire hierarchy of biological information. These tools and approaches could be mobilized to understand, detect and treat cancerous processes and establish methods applicable across a wide range of cancers.

Abstract

In Xenopus embryos, the cell cycle is driven by an autonomous biochemical oscillator that controls the periodic activation and inactivation of cyclin B1-CDK1. The oscillator circuit includes a system of three interlinked positive and double-negative feedback loops (CDK1 -> Cdc25 -> CDK1; CDK1 -/ Wee1 -/ CDK1; and CDK1 -/ Myt1 -/ CDK1) that collectively function as a bistable trigger. Previous work established that this bistable trigger is essential for CDK1 oscillations in the early embryonic cell cycle. Here, we assess the importance of the trigger in the somatic cell cycle, where checkpoints and additional regulatory mechanisms could render it dispensable. Our approach was to express the phosphorylation site mutant CDK1AF, which short-circuits the feedback loops, in HeLa cells, and to monitor cell cycle progression by live cell fluorescence microscopy. We found that CDK1AF-expressing cells carry out a relatively normal first mitosis, but then undergo rapid cycles of cyclin B1 accumulation and destruction at intervals of 3-6 h. During these cycles, the cells enter and exit M phase-like states without carrying out cytokinesis or karyokinesis. Phenotypically similar rapid cycles were seen in Wee1 knockdown cells. These findings show that the interplay between CDK1, Wee1/Myt1, and Cdc25 is required for the establishment of G1 phase, for the normal approximately 20-h cell cycle period, and for the switch-like oscillations in cyclin B1 abundance characteristic of the somatic cell cycle. We propose that the HeLa cell cycle is built upon an unreliable negative feedback oscillator and that the normal high reliability, slow pace and switch-like character of the cycle is imposed by a bistable CDK1/Wee1/Myt1/Cdc25 system.

Abstract

A simple negative feedback loop of interacting genes or proteins has the potential to generate sustained oscillations. However, many biological oscillators also have a positive feedback loop, raising the question of what advantages the extra loop imparts. Through computational studies, we show that it is generally difficult to adjust a negative feedback oscillator's frequency without compromising its amplitude, whereas with positive-plus-negative feedback, one can achieve a widely tunable frequency and near-constant amplitude. This tunability makes the latter design suitable for biological rhythms like heartbeats and cell cycles that need to provide a constant output over a range of frequencies. Positive-plus-negative oscillators also appear to be more robust and easier to evolve, rationalizing why they are found in contexts where an adjustable frequency is unimportant.

Abstract

microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.

Abstract

A typical protein kinase must recognize between one and a few hundred bona fide phosphorylation sites in a background of approximately 700,000 potentially phosphorylatable residues. Multiple mechanisms have evolved that contribute to this exquisite specificity, including the structure of the catalytic site, local and distal interactions between the kinase and substrate, the formation of complexes with scaffolding and adaptor proteins that spatially regulate the kinase, systems-level competition between substrates, and error-correction mechanisms. The responsibility for the recognition of substrates by protein kinases appears to be distributed among a large number of independent, imperfect specificity mechanisms.

Substrate competition as a source of ultrasensitivity in the inactivation of Wee1CELLKim, S. Y., Ferrell, J. E.2007; 128 (6): 1133-1145

Abstract

The mitotic regulators Wee1 and Cdk1 can inactivate each other through inhibitory phosphorylations. This double-negative feedback loop is part of a bistable trigger that makes the transition into mitosis abrupt and decisive. To generate a bistable response, some component of a double-negative feedback loop must exhibit an ultrasensitive response to its upstream regulator. Here, we experimentally demonstrate that Wee1 exhibits a highly ultrasensitive response to Cdk1. Several mechanisms can, in principle, give rise to ultrasensitivity, including zero-order effects, multisite phosphorylation, and competition mechanisms. We found that the ultrasensitivity in the inactivation of Wee1 arises mainly through two competition mechanisms: competition between two sets of phosphorylation sites in Wee1 and between Wee1 and other high-affinity Cdk1 targets. Based on these findings, we were able to reconstitute a highly ultrasensitive Wee1 response with purified components. Competition provides a simple way of generating the equivalent of a highly cooperative allosteric response.

Abstract

Vertebrate eggs arrest at metaphase of meiosis II due to an activity known as cytostatic factor (CSF). CSF antagonizes the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C), preventing cyclin B destruction and meiotic exit until fertilization occurs. A puzzling feature of CSF arrest is that APC/C inhibition is leaky. Ongoing cyclin B synthesis is counterbalanced by a limited amount of APC/C-mediated cyclin B destruction; thus, cyclin B/Cdc2 activity remains at steady state. How the APC/C can be slightly active toward cyclin B, and yet restrained from ubiquitinating cyclin B altogether, is unknown. Emi2/XErp1 is the critical CSF component directly responsible for APC/C inhibition during CSF arrest. Fertilization triggers the Ca2+-dependent destruction of Emi2, releasing the APC/C to ubiquitinate the full pool of cyclin B and initiate completion of meiosis. Previously, we showed that a phosphatase maintains Emi2's APC/C-inhibitory activity in CSF-arrested Xenopus egg extracts. Here, we demonstrate that phosphatase inhibition permits Emi2 phosphorylation at thr-545 and -551, which inactivates Emi2. Furthermore, we provide evidence that adding excess cyclin B to CSF extracts stimulates Cdc2 phosphorylation of these same residues, antagonizing Emi2-APC/C association. Our findings suggest a model wherein the pool of Emi2 acts analogously to a rheostat by integrating Cdc2 and phosphatase activities to prevent cyclin B overaccumulation and Cdc2 hyperactivity during the indefinite period of time between arrival at metaphase II and eventual fertilization. Finally, we propose that inactivation of Emi2 by Cdc2 permits mitotic progression during early embryonic cleavage cycles.

Abstract

Cyclin-dependent kinase activation can prevent yeast cells from responding to mating pheromone. Strickfaden et al. (2007) now show that this block arises from the multisite phosphorylation of Ste5. This provides a beautiful example of how phosphorylation can produce decisive changes in protein function through bulk electrostatics, without the necessity of intricate conformational changes.

Abstract

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.

Abstract

The protein kinase Mos is responsible for the activation of MEK1 and p42 mitogen-activated protein kinase during Xenopus oocyte maturation and during mitosis in Xenopus egg extracts. Here we show that the activation of Mos depends upon the phosphorylation of Ser 3, a residue previously implicated in the regulation of Mos stability; the dephosphorylation of Ser 105, a previously unidentified phosphorylation site conserved in Mos proteins; and the regulated dissociation of Mos from CK2beta. Mutation of Ser 3 to alanine and/or mutation of Ser 105 to glutamate produces a Mos protein that is defective for M-phase activation, as assessed by in vitro kinase assays, and defective for induction of oocyte maturation and maintenance of the spindle assembly checkpoint in extracts. Interestingly, Ser 105 is situated at the beginning of helix alphaC in the N-terminal lobe of the Mos kinase domain. Changes in the orientation of this helix have been previously implicated in the activation of Cdk2 and Src family tyrosine kinases. Our work suggests that Ser 105 dephosphorylation represents a novel mechanism for reorienting helix alphaC.

Abstract

During mitosis, a select pool of MEK1 and p42/p44 MAPK becomes activated at the kinetochores and spindle poles, without substantial activation of the bulk of the cytoplasmic p42/p44 MAPK. Recently, we set out to identify the MAP kinase kinase kinase (MAPKKK) responsible for this mitotic activation, using cyclin-treated Xenopus egg extracts as a model system, and presented evidence that Mos was the relevant MAPKKK . However, a second MAPKKK distinct from Mos was readily detectable as well. Here, we partially purify this second MAPKKK and identify it as B-Raf. No changes in the activity of B-Raf were detectable during progesterone-induced oocyte maturation, after egg fertilization, or during the early embryonic cell cycle, arguing against a role for B-Raf in the mitotic activation of MEK1 and p42 MAPK. Ras proteins can bring about activation of MEK1 and p42 MAPK in extracts, and Ras may contribute to signaling from the classical progesterone receptor during oocyte maturation and from receptor tyrosine kinases during early embryogenesis. We found that both B-Raf and C-Raf, but not Mos, are required for Ras-induced MEK1 and p42 MAPK activation. These data indicate that two upstream stimuli, active Ras and active Cdc2, utilize different MAPKKKs to activate MEK1 and p42 MAPK.

Abstract

Microarray studies have shown that individual synthetic small interfering RNAs (siRNAs) can have substantial off-target effects. Pools of siRNAs, produced by incubation of dsRNAs with recombinant Dicer or RNase III, can also be used to silence genes. Here we show that diced siRNA pools are highly complex, containing hundreds of different individual siRNAs. This high complexity could either compound the problem of off-target effects, since the number of potentially problematic siRNAs is high, or it could diminish the problem, since the concentration of any individual problematic siRNA is low. We therefore compared the off-target effects of diced siRNAs to chemically synthesized siRNAs. In agreement with previous reports, we found that two chemically synthesized siRNAs targeted against p38alpha MAPK (MAPK14) induced off-target changes in the abundance of hundreds of mRNAs. In contrast, three diced siRNA pools against p38alpha MAPK had almost no off-target effects. The off-target effects of a synthetic siRNA were reduced when the siRNA was diluted 3-fold in a diced pool and completely alleviated when it was diluted 30- or 300-fold, suggesting that when problematic siRNAs are present within a diced pool, their absolute concentration is too low to result in significant off-target effects. These data rationalize the observed high specificity of RNA interference in C. elegans and D. melanogaster, where gene suppression is mediated by endogenously-generated diced siRNA pools, and provide a strategy for improving the specificity of RNA interference experiments and screens in mammalian cells.

Abstract

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.

Abstract

The Cdk1 inhibitor Wee1 is inactivated during mitotic entry by proteolysis, translational regulation, and transcriptional regulation. Wee1 is also regulated by posttranslational modifications, and here we have identified five phosphorylation sites in the N-terminal domain of embryonic Xenopus Wee1A through a combination of mutagenesis studies and matrix-assisted laser desorption ionization-time of flight mass spectrometry. All five sites conform to the Ser-Pro/Thr-Pro consensus for proline-directed kinases like Cdks. Three of the sites (Ser 38, Thr 53, and Ser 62) are required for the mitotic gel shift, and at least two of these sites (Ser 38 and Thr 53) regulate the proteolysis of Wee1A during interphase. The other two sites (Thr 104 and Thr 150) are primarily responsible for the mitotic inactivation of Wee1A. Alanine mutants of Thr 150 or Thr 104 had an increased capacity to inhibit mitotic entry in cyclin B-treated interphase extracts, and Thr 150 was found to be transiently phosphorylated just prior to nuclear envelope breakdown in cycling egg extracts. These findings establish the phosphorylation-dependent direct inactivation of Wee1A as a critical mechanism for the promotion of M-phase entry. These results also show that multisite phosphorylation cooperatively inactivates Wee1A and cooperatively promotes Wee1A proteolysis.

Abstract

Positive feedback is a ubiquitous signal transduction motif that allows systems to convert graded inputs into decisive, all-or-none outputs. Here we investigate why the positive feedback switches that regulate polarization of budding yeast, calcium signaling, Xenopus oocyte maturation, and various other processes use multiple interlinked loops rather than single positive feedback loops. Mathematical simulations revealed that linking fast and slow positive feedback loops creates a "dual-time" switch that is both rapidly inducible and resistant to noise in the upstream signaling system.

Abstract

The cell-cycle oscillator includes an essential negative-feedback loop: Cdc2 activates the anaphase-promoting complex (APC), which leads to cyclin destruction and Cdc2 inactivation. Under some circumstances, a negative-feedback loop is sufficient to generate sustained oscillations. However, the Cdc2/APC system also includes positive-feedback loops, whose functional importance we now assess. We show that short-circuiting positive feedback makes the oscillations in Cdc2 activity faster, less temporally abrupt, and damped. This compromises the activation of cyclin destruction and interferes with mitotic exit and DNA replication. This work demonstrates a systems-level role for positive-feedback loops in the embryonic cell cycle and provides an example of how oscillations can emerge out of combinations of subcircuits whose individual behaviors are not oscillatory. This work also underscores the fundamental similarity of cell-cycle oscillations in embryos to repetitive action potentials in pacemaker neurons, with both systems relying on a combination of negative and positive-feedback loops.

Abstract

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.

Abstract

Aberrant accumulation of extensively phosphorylated heavy (high molecular weight) neurofilament (NFH) and neurodegeneration are features of hereditary canine spinal muscular atrophy (HCSMA), an animal model of human motor neuron disease. In this study, the canine NFH gene was mapped, cloned, and sequenced, and electrospray/mass spectrometry was used to evaluate the phosphorylation state of NFH protein from normal dogs and dogs with HCSMA. The canine NFH gene was localized to a region on canine chromosome 26 that corresponds to human NFH on chromosome 22q. The predicted length of the canine NFH protein is 1135 amino acids, and it shares an 80.3% identity with human NFH and >74.6% with murine NFH proteins. Direct sequencing of NFH cDNA from HCSMA dogs revealed no mutations, although cDNA sequence and restriction fragment length polymorphism (RFLP) analysis indicates that there are at least three canine NFH alleles, differing in the position and number (61 or 62) of Lys-Ser-Proline (KSP) motifs. The two longest alleles (L1 and L2), each with 62 KSP repeats, contain an additional 24-base insert and were observed in both normal and HCSMA dogs. However, the shorter allele (the C allele), with 61 KSP sites and lacking the 24-base insertion, was absent in dogs with HCSMA. Mass spectrometry data indicated that almost all of the NFH KSP phosphorylation sites were occupied. No new or extra sites were identified in native NFH purified from the HCSMA dogs. The predominance of the two longest NFH alleles and the additional KSP phosphorylation sites they confer probably account for the presence of extensively phosphorylated NFs detected immunohistochemically in dogs with HCSMA.

Abstract

In addition to their activation via binding to cyclins, cyclin-dependent kinases (CDKs) can be activated via binding to a novel cell cycle regulator termed Speedy/Ringo, which shows no apparent similarity to cyclins. The first Speedy/Ringo protein was found to be essential for Xenopus oocyte maturation and a human homolog (Spy1, herein called Speedy/ Ringo A1) regulates S-phase entry and cell survival after DNA damage in cultured somatic cells. We have identified a Speedy/Ringo-like gene in the most primitive branching clade of chordates (Ciona intestinalis), as well as four mammalian homologs. Of the mammalian proteins, two, Speedy/Ringo A and C, bind to Cdc2 and Cdk2, whereas Speedy/Ringo B binds preferentially to Cdc2. Despite their distinct CDK-binding preferences, both Speedy/Ringo A and B can promote the maturation of Xenopus oocytes and all three Speedy/Ringo proteins can bind to and activate CDKs in vivo. These mammalian Speedy/Ringo proteins exhibit distinct tissue expression patterns, though all three are enriched in testis, consistent with the initial observation that Xenopus Speedy/Ringo functions during meiosis. Speedy/Ringo A is widely expressed in tissues and cell lines. Speedy/Ringo B expression appears to be testis-specific. Speedy/Ringo C is expressed in diverse tissues, particularly those that undergo polyploidization. All Speedy/Ringo proteins share a highly conserved approximately 140-aa domain we term the Speedy/Ringo box that is essential for CDK binding. Point mutations in this domain abolish CDK binding. Besides the central Speedy/Ringo box, Speedy/Ringo A contains a C-terminal portion, which promotes CDK activation, and an N-terminal portion, which is dispersible for both CDK binding and activation but that influences protein expression. The existence of this growing family of CDK activators suggests that Speedy/Ringo proteins may play as complex a role in cell cycle control as the diverse family of cyclins.

Abstract

The ERK1/ERK2 MAP kinases (MAPKs) are transiently activated during mitosis, and MAPK activation has been implicated in the spindle assembly checkpoint and in establishing the timing of an unperturbed mitosis. The MAPK activator MEK1 is required for mitotic activation of p42 MAPK in Xenopus egg extracts; however, the identity of the kinase that activates MEK1 is unknown. Here we have partially purified a Cdc2-cyclin B-induced MEK-activating protein kinase from mitotic Xenopus egg extracts and identified it as the Mos protooncoprotein, a MAP kinase kinase kinase present at low levels in mitotic egg extracts, early embryos, and somatic cells. Immunodepletion of Mos from interphase egg extracts was found to abolish Delta90 cyclin B-Cdc2-stimulated p42 MAPK activation. In contrast, immunodepletion of Raf-1 and B-Raf, two other MEK-activating kinases present in Xenopus egg extracts, had little effect on cyclin-stimulated p42 MAPK activation. Immunodepletion of Mos also abolished the transient activation of p42 MAPK in cycling egg extracts. Taken together, these data demonstrate that Mos is responsible for the mitotic activation of the p42 MAPK pathway in Xenopus egg extracts.

Abstract

Recent work has shown that the efficacy of a small interfering RNA (siRNA) for silencing gene expression is a function of how easy it is to unwind the siRNA from the 5'-antisense end. Based on these insights, one group has designed an algorithm that substantially improves the odds of picking an effective siRNA, and two groups have shown that 'forked' or 'frayed' siRNAs, which should be easier to unwind from the 5'-antisense end, are more effective than conventional siRNAs. These strategies represent important steps towards the rational design of effective siRNAs.

Abstract

Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.

Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feed back systemsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAAngeli, D., Ferrell, J. E., Sontag, E. D.2004; 101 (7): 1822-1827

Abstract

It is becoming increasingly clear that bistability (or, more generally, multistability) is an important recurring theme in cell signaling. Bistability may be of particular relevance to biological systems that switch between discrete states, generate oscillatory responses, or "remember" transitory stimuli. Standard mathematical methods allow the detection of bistability in some very simple feedback systems (systems with one or two proteins or genes that either activate each other or inhibit each other), but realistic depictions of signal transduction networks are invariably much more complex. Here, we show that for a class of feedback systems of arbitrary order the stability properties of the system can be deduced mathematically from how the system behaves when feedback is blocked. Provided that this open-loop, feedback-blocked system is monotone and possesses a sigmoidal characteristic, the system is guaranteed to be bistable for some range of feedback strengths. We present a simple graphical method for deducing the stability behavior and bifurcation diagrams for such systems and illustrate the method with two examples taken from recent experimental studies of bistable systems: a two-variable Cdc2/Wee1 system and a more complicated five-variable mitogen-activated protein kinase cascade.

Abstract

The maturation of Xenopus oocytes can be thought of as a process of cell fate induction, with the immature oocyte representing the default fate and the mature oocyte representing the induced fate. Crucial mediators of Xenopus oocyte maturation, including the p42 mitogen-activated protein kinase (MAPK) and the cell-division cycle protein kinase Cdc2, are known to be organized into positive feedback loops. In principle, such positive feedback loops could produce an actively maintained 'memory' of a transient inductive stimulus and could explain the irreversibility of maturation. Here we show that the p42 MAPK and Cdc2 system normally generates an irreversible biochemical response from a transient stimulus, but the response becomes transient when positive feedback is blocked. Our results explain how a group of intrinsically reversible signal transducers can generate an irreversible response at a systems level, and show how a cell fate can be maintained by a self-sustaining pattern of protein kinase activation.

Abstract

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.

Abstract

In the early embryonic cell cycle, Cdc2-cyclin B functions like an autonomous oscillator, whose robust biochemical rhythm continues even when DNA replication or mitosis is blocked. At the core of the oscillator is a negative feedback loop; cyclins accumulate and produce active mitotic Cdc2-cyclin B; Cdc2 activates the anaphase-promoting complex (APC); the APC then promotes cyclin degradation and resets Cdc2 to its inactive, interphase state. Cdc2 regulation also involves positive feedback, with active Cdc2-cyclin B stimulating its activator Cdc25 (refs 5-7) and inactivating its inhibitors Wee1 and Myt1 (refs 8-11). Under the correct circumstances, these positive feedback loops could function as a bistable trigger for mitosis, and oscillators with bistable triggers may be particularly relevant to biological applications such as cell cycle regulation. Therefore, we examined whether Cdc2 activation is bistable. We confirm that the response of Cdc2 to non-degradable cyclin B is temporally abrupt and switch-like, as would be expected if Cdc2 activation were bistable. We also show that Cdc2 activation exhibits hysteresis, a property of bistable systems with particular relevance to biochemical oscillators. These findings help establish the basic systems-level logic of the mitotic oscillator.

Abstract

RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.

Abstract

JNK proteins are ubiquitously expressed, evolutionarily conserved MAP kinases that are involved in stress responses. Recently, it was shown that the JNK cascade in Xenopus oocytes exhibits sustained, all-or-none responses to graded, transient stimuli. Here, we have examined the character of the JNK cascade's response in mammalian cells. The steady-state responses of JNK to sorbitol and anisomycin were found to be highly ultrasensitive in HeLa cells, HEK 293 cells, and Jurkat T cells. The JNK responses were also reversible, not sustained, as was the case in oocytes. Jurkat cells activated their JNK in response to phorbol myristate acetate (PMA), and the response of the entire population of Jurkat cells was graded. However, analysis of subpopulations of the PMA-treated Jurkat cells revealed that the steady-state responses of both JNK and CD69, a T cell surface activation marker, were essentially all-or-none in character. These studies show that the JNK cascade commonly exhibits switch-like responses to a variety of stimuli.

Abstract

Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore the fundamental importance of proximity in enzyme regulation and of keeping a proper distance for maintaining signaling specificity.

Abstract

Dual-specificity protein phosphatases are implicated in the direct down-regulation of mitogen-activated protein kinase (MAPK) activity in vivo. Accumulating evidence suggests that these phosphatases are components of negative feedback loops that restore MAPK activity to low levels after diverse physiological responses. Limited information exists, however, regarding their posttranscriptional regulation. We cloned two Xenopus homologs of the mammalian dual-specificity MAPK phosphatases MKP-1/CL100 and found that overexpression of XCL100 in G2-arrested oocytes delayed or prevented progesterone-induced meiotic maturation. Epitope-tagged XCL100 was phosphorylated on serine during G2 phase, and on serine and threonine in a p42 MAPK-dependent manner during M phase. Threonine phosphorylation mapped to a single residue, threonine 168. Phosphorylation of XCL100 had no measurable effect on its ability to dephosphorylate p42 MAPK. Similarly, mutation of threonine 168 to either valine or glutamate did not significantly alter the binding affinity of a catalytically inactive XCL100 protein for active p42 MAPK in vivo. XCL100 was a labile protein in G2-arrested and progesterone-stimulated oocytes; surprisingly, its degradation rate was increased more than twofold after exposure to hyperosmolar sorbitol. In sorbitol-treated oocytes expressing a conditionally active DeltaRaf-DD:ER chimera, activation of the p42 MAPK cascade led to phosphorylation of XCL100 and a pronounced decrease in the rate of its degradation. Our results provide mechanistic insight into the regulation of a dual-specificity MAPK phosphatase during meiotic maturation and the adaptation to cellular stress.

Abstract

Remarkably, SCF(Cdc4) ubiquitin ligase binds and ubiquitinates Sic1 decorated with six, but not five, phosphates. This numerical wizardry suggests how analog inputs can be rectified to digital outputs. Unraveling the counting mechanism promises to generate new insights into the architecture of protein nanoprocessors.

Abstract

The induction of Xenopus laevis oocyte maturation by progesterone is a striking example of a steroid hormone-mediated event that does not require transcription. Here we have investigated the role of the classical progesterone receptor in this nongenomic signaling. The Xenopus progesterone receptor (XPR) was predominantly cytoplasmic; however, a significant fraction ( approximately 5%) of one form of the receptor (p82 XPR) was associated with the plasma membrane-containing P-10,000 fraction, compatible with the observation that membrane-impermeant derivatives of progesterone can induce maturation. XPR co-precipitated with active phosphatidylinositol 3-kinase. The phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin delayed progesterone-induced maturation and completely blocked the insulin-dependent maturation, indicating that the association of XPR with PI3-K could be functionally important. We also examined whether the nongenomic signaling properties of XPR can account for the ability of glucocorticoids and the progesterone antagonist RU486 to induce maturation. We found that none of these steroids cause XPR to become associated with active PI3-K; thus, association of XPR with active PI3-K is progesterone-specific. Finally, we showed that p42 mitogen-activated protein kinase (MAPK) associates with XPR after progesterone-induced germinal vesicle breakdown and that active recombinant MAPK is able to phosphorylate p110 XPR in vitro. These findings demonstrate that the classical progesterone receptor is involved in progesterone-induced nongenomic signaling in Xenopus oocytes and provide evidence that p42 MAPK and PI3-K activity are directly associated with the classical progesterone receptor.

Abstract

Important signaling properties, like adaptation, oscillations, and bistability, can emerge at the level of relatively simple systems of signaling proteins. Here, we have examined the quantitative properties of one well-studied signaling system, the JNK cascade. We experimentally assessed the response of JNK to a physiological stimulus (progesterone) and a pathological stress (hyperosmolar sorbitol) in Xenopus laevis oocytes, a cell type that is well-suited to the quantitative analysis of cell signaling. Our aim was to determine whether JNK responses are graded (Michaelian) in character; ultrasensitive in character, resembling the responses of cooperative enzymes; or bistable and all-or-none in character.The responses of JNK to both progesterone and sorbitol were found to be essentially all-or-none. Individual oocytes had either very high or very low JNK activities, with few oocytes possessing intermediate levels of JNK activity. Moreover, JNK activation was autocatalytic, indicating that the JNK cascade is either embedded in or downstream of a positive feedback loop. JNK also exhibited hysteresis, a form of biochemical memory, in its response to sorbitol. These findings indicate that the JNK cascade is part of a bistable signaling system in oocytes.In Xenopus oocytes, JNK responds to physiological and pathological stimuli in an all-or-none manner. The JNK response shows all the hallmarks of a bistable response, including strong positive feedback and hysteresis. Bistability is a recurring theme in the biochemistry of oocyte maturation and early embryogenesis; the Mos/MEK/p42 MAPK cascade also exhibits bistable responses, and the Cdc2/cyclin B system is hypothesized to be bistable as well. However, the mechanisms underpinning the positive feedback and bistability in the three cases are different, suggesting that evolution has repeatedly converged upon bistability as a way of producing digital responses.

Abstract

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family that play critical roles in stress responses and apoptosis. We have discovered that JNK is present in Xenopus oocytes, an experimental system that offers a variety of powerful experimental approaches to questions of protein function and regulation. Like ERK2/p42 MAPK, JNK is activated just prior to germinal vesicle breakdown during Xenopus oocyte maturation and remains active throughout meiosis I and II. However, unlike p42 MAPK, which is inactivated about 30 min after eggs are fertilized or parthenogenetically activated, JNK stays constitutively active until the early gastrula stage of embryogenesis. These findings suggest that the JNK pathway may play a role in oocyte maturation and embryogenesis. JNK was activated by microinjection of Mos, by activation of an estrogen-inducible form of Raf, and by a constitutively active MEK-1 (MEK R4F), indicating that the p42 MAPK cascade can trigger JNK activation. However, the MEK inhibitor U0126 blocked progesterone-induced p42 MAPK activation but not progesterone-induced JNK activation. Thus, progesterone can stimulate JNK activation both through the MEK/p42 MAPK pathway and through MEK/p42 MAPK-independent pathways. Many of the key substrates of JNKs identified to date are transcriptional regulators. However, since transcription is not required for germinal vesicle breakdown in progesterone-treated oocytes or for the early embryonic cell cycles, our findings suggest that in these contexts the JNK pathway exerts nongenomic effects.

Abstract

Xenopus oocyte maturation is an example of an all-or-none, irreversible cell fate induction process. In response to a submaximal concentration of the steroid hormone progesterone, a given oocyte may either mature or not mature, but it can exist in intermediate states only transiently. Moreover, once an oocyte has matured, it will remain arrested in the mature state even after the progesterone is removed. It has been hypothesized that the all-or-none character of oocyte maturation, and some aspects of the irreversibility of maturation, arise out of the bistability of the signal transduction system that triggers maturation. The bistability, in turn, is hypothesized to arise from the way the signal transducers are organized into a signaling circuit that includes positive feedback (which makes it so that the system cannot rest in intermediate states) and ultrasensitivity (which filters small stimuli out of the feedback loop, allowing the system to have a stable off-state). Here we review two simple graphical methods that are commonly used to analyze bistable systems, discuss the experimental evidence for bistability in oocyte maturation, and suggest that bistability may be a common means of producing all-or-none responses and a type of biochemical memory. (c) 2001 American Institute of Physics.

Abstract

The fast-growing nontuberculous mycobacterial species Mycobacterium chelonae was isolated from six captive South African clawed frogs (Xenopus laevis) with chronic weight loss and nonhealing ulcerative skin lesions. Three of the M. chelonae isolates were evaluated to confirm the species identification using polymerase chain reaction restriction analysis. Disease associated with M. chelonae is reported mainly in people and in fish. To our knowledge, this is the first report of disease associated with M. chelonae in a colony of captive Xenopus sp.

Abstract

The 90-kDa ribosomal S6 kinases, the p90 Rsks, are a family of intracellular serine/threonine protein kinases distinguished by two distinct kinase domains. Rsks are activated downstream of the ERK1 (p44) and ERK2 (p42) mitogen-activated protein (MAP) kinases in diverse biological contexts, including progression through meiotic and mitotic M phases in Xenopus oocytes and cycling Xenopus egg extracts, and are critical for the M phase functions of Xenopus p42 MAPK. Here we report the cloning and biochemical characterization of Xenopus Rsk2. Xenopus Rsk1 and Rsk2 are specifically recognized by commercially available RSK1 and RSK2 antisera on immunoblots, but both Rsk1 and Rsk2 are immunoprecipitated by RSK1, RSK2, and RSK3 sera. Rsk2 is about 20-fold more abundant than the previously described Xenopus Rsk1 protein; their concentrations are approximately 120 and 5 nm, respectively. Rsk2, like Rsk1, forms a heteromeric complex with p42 MAP kinase. This interaction depends on sequences at the extreme C terminus of Rsk2 and can be disrupted by a synthetic peptide derived from the C-terminal 20 amino acids of Rsk2. Finally, we demonstrate that p42 MAP kinase can activate recombinant Rsk2 in vitro to a specific activity comparable to that found in Rsk2 that has been activated maximally in vivo. These findings underscore the importance of the Rsk2 isozyme in the M phase functions of p42 MAP kinase and provide tools for further examining Rsk2 function.

Abstract

Scaffold proteins play an important role in coordinating signal transduction cascades. However, their exact mechanism of action and the ultimate effect they have on the signal output remain unclear. Ferrell discusses how computer simulations have provided insight into the multiple possible functions that scaffold proteins may have. What remains is to test the predictions in real cells to determine what difference the presence of a scaffold really makes in the output of a signaling pathway.

Abstract

Xenopus oocytes and eggs provide a dramatic example of how the consequences of p42 mitogen-activated protein kinase (p42 MAPK) activation depend on the particular context in which the activation occurs. In oocytes, the activation of Mos, MEK, and p42 MAPK is required for progesterone-induced Cdc2 activation, and activated forms of any of these proteins can bring about Cdc2 activation in the absence of progesterone. However, in fertilized eggs, activation of the Mos/MEK/p42 MAPK pathway has the opposite effect, inhibiting Cdc2 activation and causing a G2 phase delay or arrest. In the present study, we have investigated the mechanism and physiological significance of the p42 MAPK-induced G2 phase arrest, using Xenopus egg extracts as a model system. We found that Wee1-depleted extracts were unable to arrest in G2 phase in response to Mos, and adding back Wee1 to the extracts restored their ability to arrest. This finding formally places Wee1 downstream of Mos/MEK/p42 MAPK. Purified recombinant p42 MAPK was found to phosphorylate recombinant Wee1 in vitro at sites that are phosphorylated in extracts. Phosphorylation by p42 MAPK resulted in a modest ( approximately 2-fold) increase in the kinase activity of Wee1 toward Cdc2. Titration experiments in extracts demonstrated that a twofold increase in Wee1 activity is sufficient to cause the delay in mitotic entry seen in Mos-treated extracts. Finally, we present evidence that the negative regulation of Cdc2 by Mos/MEK/p42 MAPK contributes to the presence of an unusually long G2 phase in the first mitotic cell cycle. Prematurely inactivating p42 MAPK in egg extracts resulted in a corresponding hastening of the first mitosis. The negative effect of p42 MAPK on Cdc2 activation may help ensure that the first mitotic cell cycle is long enough to allow karyogamy to be accomplished successfully.

Abstract

Xenopus oocyte maturation requires the phosphorylation and activation of p42 mitogen-activated protein kinase (MAPK). Likewise, the dephosphorylation and inactivation of p42 MAPK are critical for the progression of fertilized eggs out of meiosis and through the first mitotic cell cycle. Whereas the kinase responsible for p42 MAPK activation is well characterized, little is known concerning the phosphatases that inactivate p42 MAPK. We designed a microinjection-based assay to examine the mechanism of p42 MAPK dephosphorylation in intact oocytes. We found that p42 MAPK inactivation is mediated by at least two distinct phosphatases, an unidentified tyrosine phosphatase and a protein phosphatase 2A-like threonine phosphatase. The rates of tyrosine and threonine dephosphorylation were high and remained constant throughout meiosis, indicating that the dramatic changes in p42 MAPK activity seen during meiosis are primarily attributable to changes in MAPK kinase activity. The overall control of p42 MAPK dephosphorylation was shared among four partially rate-determining dephosphorylation reactions, with the initial tyrosine dephosphorylation of p42 MAPK being the most critical of the four. Our findings provide biochemical and kinetic insight into the physiological mechanism of p42 MAPK inactivation.

Building a cellular switch: more lessons from a good eggBIOESSAYSFerrell, J. E.1999; 21 (10): 866-870

Abstract

Xenopus oocytes mature in response to the steroid hormone progesterone. At the level of a population of oocytes, the response is graded-the higher the concentration of progesterone, the larger the fraction of oocytes that will mature-but at the level of the individual oocyte, the response is all-or-none. The all-or-none character of this cell fate switch is hypothesized to arise out of two properties of the signal transduction machinery that mediates maturation, positive feedback, and ultrasensitivity. This combination of positive feedback plus ultrasensitivity crops up again and again in cellular switches, from the lysis-lysogeny switch in phage-infected bacteria to the action potential in neurons.

Abstract

Fully grown Xenopus oocytes can remain in their immature state essentially indefinitely, or, in response to the steroid hormone progesterone, can be induced to develop into fertilizable eggs. This process is termed oocyte maturation. Oocyte maturation is initiated by a novel plasma membrane steroid hormone receptor. Progesterone brings about inhibition of adenylate cyclase and activation of the Mos/MEK1/p42 MAP kinase cascade, which ultimately brings about the activation of the universal M phase trigger Cdc2/cyclin B. Oocyte maturation provides an interesting example of how signaling cascades entrain the cell cycle clock to environmental changes.

Abstract

During the summer of 1996, an outbreak of Flavobacterium meningosepticum infection developed in a colony of South African clawed frogs (Xenopus laevis). Clinical signs were consistent with septicemia: ascites, anasarca, dyspnea, extreme lethargy, congestion of web vessels, petechial hemorrhages, and sudden death. Mortality rate reached 35%, and all infections were fatal. The organism was resistant to most antibiotics but was susceptible to enrofloxacin, chloramphenicol, and trimethoprim-sulfadiazine. Treatment with trimethoprim-sulfadiazine was unsuccessful. Although the point source of the infection was not determined, several environmental reservoirs were identified, including a communal water barrel and various pieces of equipment. Molecular strain typing by pulsed-field gel electrophoresis and biochemical analyses revealed that frogs were infected with a single strain of F meningosepticum. Sanitation and management procedures were effective in controlling the outbreak.

Abstract

To understand how the dramatic cell biological changes of oocyte maturation are brought about, we have begun to identify proteins whose phosphorylation state changes during Xenopus oocyte maturation. Here we have focused on one such protein, p83. We partially purified p83, obtained peptide sequence, and identified it as the intermediate chain of cytoplasmic dynein. During oocyte maturation, dynein intermediate chain became hyperphosphorylated at the time of germinal vesicle breakdown and remained hyperphosphorylated throughout the rest of meiosis and early embryogenesis. p150(Glued), a subunit of dynactin that has been shown to bind to dynein intermediate chain, underwent similar changes in its phosphorylation. Both dynein intermediate chain and p150(Glued) also became hyperphosphorylated during M phase in XTC-2 cells and HeLa cells. Thus, two components of the dynein-dynactin complex undergo coordinated phosphorylation changes at two G2/M transitions (maturation in oocytes and mitosis in cells in culture) but remain constitutively in their M phase forms during early embryogenesis. Dynein intermediate chain and p150(Glued) phosphorylation may positively regulate mitotic processes, such as spindle assembly or orientation, or negatively regulate interphase processes such as minus-end-directed organelle trafficking.

Abstract

It is widely appreciated that the regulated translocation of signaling proteins can increase the specificity and speed of signal transduction. It is less obvious that regulated translocation can also, in principle, turn a graded response into a more switch-like one. For example, if two or more signaling proteins are induced to translocate, the result can be a switch-like, ultrasensitive response. A switch-like response will also occur if translocation raises the local concentration of a signaling protein sufficiently to partially saturate the enzyme that inactivates it. These mechanisms are likely to make the mitotic activation of CDC2 (which is accompanied by the nuclear translocation of both CDC2-cyclin-B1 and its activator, CDC25C) and the growth-factor-induced activation of MAP kinase (which, upon sustained activation, concentrates in the nucleus and might thereby partially saturate the relevant MAP-kinase phosphatases) more switch-like.

Abstract

The p42 mitogen-activated protein kinase (MAPK) is required for progression through meiotic M phase in Xenopus oocytes. This report examines whether it also plays a role in normal mitotic progression. MAPK was transiently activated during mitosis in cycling Xenopus egg extracts after activation of the cyclin-dependent kinase Cdc2-cyclin B. Interference with MAPK activation by immunodepletion of its activator MEK, or by addition of the MEK inhibitor PD98059, caused precocious termination of mitosis and interfered with production of normal mitotic microtubules. Sustained activation of MAPK arrested extracts in mitosis in the absence of active Cdc2-cyclin B. These findings identify a role for MEK and MAPK in maintaining the mitotic state.

Abstract

Xenopus oocytes convert a continuously variable stimulus, the concentration of the maturation-inducing hormone progesterone, into an all-or-none biological response-oocyte maturation. Here evidence is presented that the all-or-none character of the response is generated by the mitogen-activated protein kinase (MAPK) cascade. Analysis of individual oocytes showed that the response of MAPK to progesterone or Mos was equivalent to that of a cooperative enzyme with a Hill coefficient of at least 35, more than 10 times the Hill coefficient for the binding of oxygen to hemoglobin. The response can be accounted for by the intrinsic ultrasensitivity of the oocyte's MAPK cascade and a positive feedback loop in which the cascade is embedded. These findings provide a biochemical rationale for the all-or-none character of this cell fate switch.

Abstract

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G2-phase during the first mitotic cell cycle.

Abstract

Previous work on the responses of mitogen-activated protein (MAP) kinase cascade components in a Xenopus oocyte extract system demonstrated that p42 MAP kinase (MAPK) exhibits a sharp, sigmoidal stimulus/response curve, rather than a more typical hyperbolic curve. One plausible explanation for this behavior requires the assumption that MAP kinase kinase (MAPKK) carries out its dual phosphorylation of p42 MAPK by a distributive mechanism, where MAPKK dissociates from MAPK between the first and second phosphorylations, rather than a processive mechanism, where MAPKK carries out both phosphorylations before dissociating. Here we have investigated the mechanism through which a constitutively active form of human MAPKK-1 (denoted MAPKK-1 R4F or MAPKK-1*) phosphorylates Xenopus p42 MAPK in vitro. We found that the amount of monophosphorylated MAPK formed during the phosphorylation reaction exceeded the amount of MAPKK-1* present, which would not be possible if the phosphorylation occurred exclusively by a processive mechanism. The monophosphorylated MAPK was phosphorylated predominantly on tyrosine, but a small proportion was phosphorylated on threonine, indicating that the first phosphorylation is usually, but not invariably, the tyrosine phosphorylation. We also found that the rate at which pulse-labeled monophosphorylated MAPK became bisphosphorylated depended on the MAPKK-1* concentration, behavior that is predicted by the distributive model but incompatible with the processive model. These findings indicate that MAPKK-1* phosphorylates p42 MAPK by a two-collision, distributive mechanism rather than a single-collision, processive mechanism, and provide a mechanistic basis for understanding how MAP kinase can convert graded inputs into switch-like outputs.

Abstract

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

Abstract

Recent experimental work has shown that the mitogen-activated protein (MAP) kinase cascade can convert graded inputs into switch-like outputs. The cascade could therefore filter out noise (signals of insufficient magnitude or duration) and still respond decisively to supra-threshold stimuli. Here, we explore the biochemical mechanisms likely to be at the root of this behavior.

Ultrasensitivity in the mitogen-activated protein kinase cascadePROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAHuang, C. Y., Ferrell, J. E.1996; 93 (19): 10078-10083

Abstract

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved series of three protein kinases implicated in diverse biological processes. Here we demonstrate that the cascade arrangement has unexpected consequences for the dynamics of MAPK signaling. We solved the rate equations for the cascade numerically and found that MAPK is predicted to behave like a highly cooperative enzyme, even though it was not assumed that any of the enzymes in the cascade were regulated cooperatively. Measurements of MAPK activation in Xenopus oocyte extracts confirmed this prediction. The stimulus/response curve of the MAPK was found to be as steep as that of a cooperative enzyme with a Hill coefficient of 4-5, well in excess of that of the classical allosteric protein hemoglobin. The shape of the MAPK stimulus/ response curve may make the cascade particularly appropriate for mediating processes like mitogenesis, cell fate induction, and oocyte maturation, where a cell switches from one discrete state to another.

Abstract

The progression of G2-arrested Xenopus laevis oocytes into meiotic M-phase is accompanied by the nearly simultaneous activation of p42 MAP kinase and Cdc2/cyclin B. This timing raises the possibility that the activation of one kinase might depend upon the other. Here we have examined whether Cdc2 activation requires p42 MAP kinase function. We have reconstituted Mos-induced Cdc2 activation in cell-free Xenopus oocyte extracts, and have found that Mos-induced Cdc2 activation requires active p42 MAP kinase, is inhibited by a MAP kinase phosphatase and is independent of protein synthesis. These findings indicate that p42 MAP kinase is an essential component of the M phase trigger in this system.

EVIDENCE THAT INACTIVE P42 MITOGEN-ACTIVATED PROTEIN-KINASE AND INACTIVE RSK EXIST AS A HETERODIMER IN-VIVOPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAHsiao, K. M., Chou, S. Y., Shih, S. J., Ferrell, J. E.1994; 91 (12): 5480-5484

Abstract

Mitogen-activated protein kinases (MAP kinases) are active only when phosphorylated. Here we examine whether the activation of Xenopus p42 MAP kinase might involve changes in its association with other proteins as well as changes in its phosphorylation state. We find that when p42 MAP kinase is phosphorylated and active, it is monomeric, and that when p42 MAP kinase is nonphosphorylated and inactive, about half of it is monomeric and half is a component of a 110-kDa complex. We identify Rsk, an 82-kDa protein kinase that can be phosphorylated and partially activated by p42 MAP kinase, as being specifically associated with inactive p42 MAP kinase. It is possible that the complex of inactive p42 MAP kinase and inactive Rsk acts as a single signal reception particle and that the activation of the two kinases may be better described as a fork in a bifurcating signal transduction pathway than as successive levels in a kinase cascade.

Abstract

Here we demonstrate that partially purified Xenopus p42 mitogen-activated protein (MAP) kinase phosphorylates bacterially expressed human c-Jun at a single site, serine 243. Several lines of evidence argue that this phosphorylation is due to p42 MAP kinase itself rather than some contaminating species. Phosphorylation of serine 243 markedly decreases the binding of c-Jun to oligonucleotides containing the 12-O-tetradecanoylphorbol-13-acetate response element. These findings suggest that MAP kinase may play a role in the down-regulation of c-Jun or in the cycle of transcriptional initiation and elongation.

Abstract

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.

Abstract

We have studied the intracellular distribution of the proto-oncogene product pp60c-src in human platelets by immunofluorescence microscopy and by immunogold electron microscopy on frozen thin sections. Virtually all of the immunoreactive pp60c-src was found to be associated with the plasma membrane and with membranes of the surface-connected canalicular system. This localization suggests that pp60c-src may relay signals from an as yet unidentified cell surface protein to intracellular targets.

Abstract

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.

Abstract

We have used a renaturation method to search for previously unidentified protein kinases in human platelets. The method involves subjecting lysates to denaturing gel electrophoresis, transferring the proteins to blotting membranes, and treating the blotted proteins with guanidine. The guanidine is then removed to allow the proteins to renature, and the blots are overlaid with [gamma-32P]ATP. We have identified 14 electrophoretically distinct, serine/threonine-specific protein kinases. Eleven of the kinases clearly differ in molecular weight from all previously described platelet serine/threonine kinases. Ten of these novel kinases (PK220, PK200, PK170, PK150, PK64, PK60, PK56, PK52, PK48, and PK40) were found to possess markedly increased in vitro activity when isolated from thrombin-stimulated platelets, presumably as a result of thrombin-stimulated covalent modification. Treatment of intact platelets with the calcium ionophore ionomycin and phorbol 12-myristate 13-acetate also increased the in vitro activity of these kinases. The agonist-stimulated kinases could be divided into three classes: 1) one kinase whose activity was increased by in vivo phorbol ester treatment but not by ionomycin (PK150); 2) two kinases whose activity was increased by ionomycin but not phorbol ester (PK48 and PK40); 3) seven kinases whose activity was markedly increased by combinations of phorbol ester and ionomycin, but not by either agent alone (PK220, PK200, PK170, PK64, PK60, PK56, and PK52). This third mode of regulation is what would be expected of enzymes that mediate the biological effects of inositide-mobilizing stimuli.

TYROSINE-SPECIFIC PROTEIN-PHOSPHORYLATION IS REGULATED BY GLYCOPROTEIN-IIB-IIIA IN PLATELETSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAFerrell, J. E., Martin, G. S.1989; 86 (7): 2234-2238

Abstract

We have previously shown that a number of platelet proteins become phosphorylated at tyrosine residues in response to platelet-activating agents. Here we present two lines of evidence implicating a platelet integrin, glycoprotein IIb-IIIa, in the regulation of a specific subset of these tyrosine phosphorylations. (i) Two peptides that inhibit the binding of fibrinogen and other ligands to gpIIb-IIIa, Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, also inhibited the thrombin-induced tyrosine phosphorylation of this subset of proteins. The tetrapeptide Arg-Gly-Glu-Ser, which does not inhibit fibrinogen binding, did not inhibit thrombin-stimulated tyrosine phosphorylation. (ii) Platelets lacking gpIIb-IIIa (from a subject with Glanzmann thrombasthenia) did not undergo this subset of tyrosine phosphorylation in response to thrombin, although other serine, threonine, and tyrosine phosphorylations proceeded normally. These findings suggest a role for tyrosine-specific protein phosphorylation in integrin-mediated cell-matrix recognition.

Abstract

Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp60c-src, primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60c-src function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets.

Abstract

Activated platelets adopt a characteristic spiculate morphology. A wide variety of anionic and zwitterionic amphipathic compounds were found to effect a similar shape change and to cause the open canalicular system to become less prominent. Several cationic amphipaths reversed thrombin-, PAF-, and amphipath-induced spiculation and restored the discoid shape. Higher concentrations of cationic amphipaths caused the cells to assume spheroid and indented forms, and caused the canalicular system to appear more prominent. Three amphipaths were studied further to address possible mechanisms underlying their morphological effects. Dilauroylphosphatidylcholine was found to induce spiculation without causing the changes in protein phosphorylation and inositide metabolism generally associated with platelet activation. Two other amphipaths, chlorpromazine (which induced sphering) and dilauroylphosphatidylserine (which caused spiculation followed by sphering) caused specific changes in protein and/or lipid phosphorylation, which may be responsible for some, but not all, of the morphological effects of these compounds. To account for these findings, we propose that platelet shape can be influenced by changes in the plasma membrane bilayer balance. Agents that bind to the membrane outer monolayer are accommodated by spiculation; those that bind to the inner monolayer are accommodated by sphering.

Abstract

A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.

Abstract

Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced-stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT-induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to-discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT.

Abstract

When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and red cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorbed to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 +/- 1) X 10(6) molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% +/- 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% +/- 0.2%.

Abstract

In the first part of this paper we have indicated how the techniques and capabilities of theoretical chemistry, together with experimental results, can be used in a mechanistic approach to structure-activity studies of toxicity. In the second part, we have illustrated how this computer-assisted approach has been used to identify and calculate causally related molecular indicators of relative carcinogenic activity in five classes of chemical carcinogens: polycyclic aromatic hydrocarbons and their methyl derivatives, aromatic amines, chloroethanes, chloroalkenes and dialkyl nitrosamines. In each class of chemicals studied, candidate molecular indicators have been identified that could be useful in predictive screening of unknown compounds. In addition, further insights into some mechanistic aspects of chemical carcinogenesis have been obtained. Finally, experiments have been suggested to both verify the usefulness of the indicators and test their mechanistic implications.

Abstract

ATP-depleted human erythrocytes lose their smooth discoid shape and adopt a spiny, crenated form. This shape change coincides with the conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol and phosphatidic acid to diacylglycerol. Both crenation and lipid dephosphorylation are accelerated by iodoacetamide, and both are reversed by nutrient supplementation. The observed changes in lipid populations should shrink the membrane inner monolayer by 0.6%, consistent with estimates of bilayer imbalance in crenated cells. These observations suggest that metabolic crenation arises from a loss of inner monolayer area secondary to the degradation of phosphatidylinositol-4,5-bisphosphate and phosphatidic acid. A related process, crenation after Ca2+ loading, appears to arise from a loss inositides by a different pathway.

Abstract

Crenation, the shape change that follows ATP depletion in human erythrocytes, also follows ionphore-mediated Ca2+-loading. Experiments designed to test whether Ca2+ mediates metabolic crenation showed that: (1) an influx of extracellular Ca2+ is not required for metabolic crenation; (2) metabolic crenation is accompanied by a 70% increase in 86Rb+ permeability, a change much smaller than the increase expected if crenating concentrations of Ca2+ were released from bound intracellular pools; (3) A23187 plus EGTA, a treatment that depletes intracellular Ca2+ and stops Ca2+ crenation, does not affect metabolic crenation; (4) calmodulin inhibitors do not slow metabolic crenation. We conclude that Ca2+ does not mediate metabolic crenation. Albumin washes reverse Ca2+ crenation and metabolic crenation involve the accumulation of some amphiphilic species (e.g., lysolipid or diacylglycerol) in the cell membrane outer monolayer, and that ATP depletion induces a second crenating process which might be a reorganization of the cytoskeleton.

Abstract

Membrane protein phosphorylation has been studied in intact human erythrocytes and in resealed erythrocyte ghosts by measuring the incorporation of 32P into band 2 of spectrin. alpha-Adrenergic agonists and Ca+2 stimulate 32P-phosphate incorporation, an effect inhibited by trifluoperazine and diminished in resealed ghosts depleted of calmodulin. Ghosts prepared with endogenous calmodulin or resealed around purified calmodulin exhibit norepinephrine- and Ca+2-stimulated phosphorylation only in the presence of [gamma-32P]-ATP. Ghosts resealed with or without calmodulin in the presence of unlabelled ATP show no net gain or loss of 32P in membrane proteins when exposed to norepinephrine or calcium stimulation. These observations suggest that calcium and norepinephrine stimulation of membrane protein phosphorylation is mediated by calmodulin-dependent spectrin kinase activity, rather than by increased turnover by spectrin ATPase or by inhibition of phosphospectrin phosphatase.

Abstract

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an alpha-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.